Aptamers are oligonucleotides that mimic antibodies in their ability to act as ligands and bind to analytes. U.S. Pat. No. 5,475,096 teaches a method for the in vitro selection of DNA or RNA molecules that are capable of binding specifically to a target molecule. This patent discloses a method of reiterative selection of oligonucleotides that has come to be known as “Systematic Evolution of Ligands by Exponential Enrichment” or SELEX. Within such a method, there is a need to separate those oligonucleotide sequences that have bound to the target from those that have not. All methods employing SELEX require a process for partitioning oligonucleotide sequences that have bound to the analyte of interest from oligonucleotide sequences that have not bound to the analyte of interest. In U.S. Pat. No. 5,475,096, this partitioning process is taught as the use of nitrocellulose membranes. Single stranded oligonucleotides will pass through nitrocellulose while many analyte molecules, in particular protein analytes, will not. Oligonucleotides that are not bound to the analyte will therefore pass through nitrocellulose, whereas oligonucleotides that are bound to the analyte will not pass through and thus can be partitioned from those that do.
The use of nitrocellulose as a means of partitioning oligonucleotides that have bound to a target analyte from oligonucleotides that have not bound to the target analyte is limited in use to those analytes that will not readily pass through nitrocellulose. This means that this is not an appropriate method for the selection of aptamers for small molecules, including but not limited to such examples as metabolites, drugs, antibiotics, peptides, and toxins, or any molecule that will pass through nitrocellulose.
In practice, we have also found difficulties with the use of this method as many proteins may pass through nitrocellulose to a certain extent.
In practice, we have also found that this method can include selection for oligonucleotide sequences that are capable of strong secondary structure and thus do not pass through nitrocellulose even though they do not bind to the target analyte. The selection of such sequences is undesirable.
U.S. Pat. No. 5,475,096 also suggests that partitioning of oligonucleotides that are bound to a target analyte from oligonucleotides that are not bound to a target analyte can also be achieved by immobilizing the target analyte within a column. This is a very common method and has been used successfully in a wide variety of applications.
The immobilization of a target analyte within a column does suffer from certain drawbacks however. One significant constraint is in regard to small molecules. The conjugation of the small molecule to a solid support in order to effect immobilization not only changes the chemical group on the small molecule used for conjugation, but also has the potential to change the entire electron charge cloud and resonance characteristics of the analyte. In such instances, it is possible to select an aptamer that binds well to the immobilized target analyte but not to the free form of the analyte. This is undesirable for diagnostic purposes when the knowledge of the quantity of the free analyte is being sought.
The immobilization of a target analyte within a column also renders it difficult to perform selection for complexes among molecules. It is an important aspect of these procedures to remove non-immobilized target from the solid support with washes. If this is not performed, then oligonucleotides may bind to a target that is subsequently lost in a wash step. This washing of the column to remove non-immobilized targets also removes any molecules that may be bound to the immobilized target in a non-covalent manner. Such complexes can be potentially rebuilt if the nature of these complexes is known. If the nature of the complex is not known however, then the complexes cannot be rebuilt.
An important part of the selection process is the washing of the bound oligonucleotides in order to increase stringency of the selection process. Weakly bound complexes will be disrupted by such washes, or may be disrupted by wash steps implicit in the conjugation process itself. Thus, this is not a sufficiently reliable method for the selection of molecular complexes.
An epitope can be defined as the region on an analyte that a ligand interacts with in terms of binding. An epitope can be a contiguous stretch within the target analyte or can be represented by multiple points that are physically proximal in a folded form of the target analyte. The immobilization of a target analyte within a column necessarily disrupts at least one epitope within the target molecule. The nature of the epitope disruption is a function of the conjugation process used. For example, conjugation relying on thiol groups on proteins will disrupt epitopes within the protein that involve cysteine residues. Such a conjugation has the potential to disrupt not only the epitope involving the residue conjugated, but also, may affect protein folding on a more global level and affect other non-proximal epitopes. This is undesirable as aptamers may be selected upon their binding to the immobilized target analyte but not to the free target analyte.
It is often desirable to obtain ligands for multiple epitopes within a target analyte. This enables the use of multiple ligands simultaneously to either capture or detect the target analyte in a diagnostic device. The removal of at least one epitope from selection is undesirable as this reduces the number of epitopes that ligands are selected for.
Mendonsa S. D. and Bowser M. T., In vitro evolution of functional DNA using capillary electrophoresis. 2004 Jan. 14. J Am. Chem. Soc. 126(1):20-1 provides a description of the use of capillary electrophoresis for the selection of aptamers against target analytes. This method does not require immobilization of either target analyte or oligonucleotides as partitioning is based on the different flow properties of the oligonucleotides bound to the target analyte as compared to both the unbound oligonucleotides and the unbound target analyte.
The method of capillary electrophoresis represents a step forward from approaches based on immobilization but still faces significant constraints. The method is not conducive for use with small molecules, as small molecules will not shift the flow of the bound oligonucleotides sufficiently from the unbound oligonucleotides.
The method of capillary electrophoresis may cause complexes among proteins or between proteins and metabolites to be disrupted by the forces of the electrical fields involved in the separation and/or the flow of the solutions. Thus, this is not a desirable selection platform for complexes.
Moreover, the method of capillary electrophoresis cannot be readily applied to complex mixtures such as blood serum, cerebral spinal fluid, urine, sweat, saliva, menstrual fluid, fecal suspensions, cell or tissue suspensions, or plant phloem solution as the identification of the bound oligonucleotides will not be possible given the complexity of the matrix.
Several other methods are known in the art such as flow cytometry, magnetic capillary electrophoresis, gel filtration, density gradient separation, and surface plasmon resonance. Each of these methods suffer from at least one of the constraints listed above.
U.S. Pat. No. 5,631,146 teaches how to use affinity chromatography to select a single stranded DNA molecule (oligonucleotide) that is capable of binding specifically to adenosine molecules.
U.S. Pat. No. 7,745,607 claims a method for the selection of aptamers which involves the use of sense and antisense pairing. Aptamer selection invariably involves the synthesis of libraries of oligonucleotides that contain known primer sequences on either end flanking a random region. U.S. Pat. No. 7,745,607 exploits this by introducing an antisense sequence for one of the known sequences flanking the random region.
The present invention is substantially different. It relies on antisense binding to the random region. This is important because it is the random region that binds to the target molecule. For U.S. Pat. No. 7,745,607 to work, there must be either an interaction between the random region and the known sense sequence, or a translational conformation shift that affects the ability of the known sense sequence capacity to bind to an antisense. In the present invention, this is not required. In the present invention, we are selecting directly for conformational changes in the random region only. This represents a larger proportion of potentially binding sequences than could be detected through the use of U.S. Pat. No. 7,745,607. This means that with the present invention, it is possible to identify more aptamers from a selection process that bind to the target molecule. The ability to select more aptamers implicitly means, on a probability basis, that it is possible to select better aptamers.
The present invention also involves selection against shapes that are not capable of binding to the immobilized antisense oligonucleotides. This reduces the potential for selection of sequence shapes that are favored by the selection process to form shapes that do not bind to the antisense sequence in the absence of any target to bind to. This can be a very significant problem for selection and is not prevented in any way by U.S. Pat. No. 7,745,607. The present invention effectively eliminates this as an issue. This means that the present invention will not mistakenly select for sequences that form certain structures in the absence of the target.
The present invention represents thus an improvement over all of these other approaches as it enables the following:                No need for immobilization of either oligonucleotide sequences or target analytes in the selection process;        Generic application to all targets regardless of size;        Capacity to modulate stringency;        Direct selection for binding with the random nucleotide region of the aptamer;        Direct selection for a shift in the shape of the aptamer;        Selection against sequences that would not bind to an antisense sequence in the absence of the target analyte.        
This invention enables several new approaches to aptamer selection that were not possible previously. Certain of these are listed below. This list is meant to be illustrative of the nature of new approaches enabled by this approach and is not meant to be exhaustive.                The ability to perform completely free/free selection enables selection for target analytes in complex mixtures such as blood serum, cerebrospinal fluid, urine, sweat, saliva, menstrual fluid, fecal suspensions, cell lysate suspensions, plant phloem fluid, ground water.        The ability to select aptamers for complexes for example including but not limited to complexes formed between proteins, or between proteins and metabolites, or between metabolites, without a risk that the immobilization process or the selection process may disrupt the complex.        The ability to select for small molecules without affecting their structure or binding capacity through conjugation.        